Thioesterases and their use

ABSTRACT

Disclosed are nucleotide sequences encoding thioesterase enzymes, methods for their production, their use in methods to form thioesters, and their use in methods of screening for other wild type bacteria capable of producing said thioesterase enzymes. 
     Also disclosed are compositions comprising thioesters produced by the methods disclosed herein.

TECHNICAL FIELD

Disclosed are nucleotide sequences encoding thioesterase enzymes,methods of their production, and their use in methods of formingthioesters.

BACKGROUND

Thioesters are important flavouring compounds for, amongst other things,cheese, vegetable, meat and coffee products. Particularly usefulthioesters include, but are not limited to, methyl butanethioate,(hereinafter “MTB”), methyl thioacetate (hereinafter “MTA”) and methylthiopropionate (hereinafter “MTP”).

Thioesters can be made synthetically. However, because of food trends,and health and wellness concerns, there is particular demand for flavourcompounds, such as thioesters, that are either obtained directly fromnatural products, or produced via biological processes. A particularadvantage of such compounds is that they may be termed “natural” andpotentially labeled as such on consumable products.

It is known that various bacteria produce thioesters duringfermentation. However, the yields and product ratios of these existingprocesses are difficult to influence.

Accordingly, it would be beneficial to develop a more predictable andpotentially more economically viable method of producing thioesters thatcould be used in consumable products and labeled as “natural”.

DETAILED DESCRIPTION

The applicant has now identified nucleotide sequences encodingthioesterase enzymes.

This finding enables the nucleotide sequences to be used in methodsenabling the efficient production of thioesters that can be added toconsumable products and labeled as “natural”. Furthermore, thesenucleotide sequences may be used in screening methods to identify otherwild type bacteria capable of forming thioesters.

According to a first illustrative embodiment, there are providednucleotide sequences, encoding enzymes having thioesterase activitycomprising the nucleotide sequences, or functional equivalents thereof,disclosed in SEQ.ID. Nos.1, 3, 5, and 7.

Without limitation, and only by way of illustration, the nucleic acidsmay be isolated from bacteria belonging to the Brevibacterium family.According to certain embodiments, the nucleic acids may be isolated frombacteria belonging to the Brevibacterium Linens species, a non limitingexamples is strain American type culture collection (herein after ATCC)9174 (BL2).

Functional equivalents of the nucleotide sequences include thosenucleotide sequences that by virtue of the degeneracy of the geneticcode possess a different nucleotide sequence to those disclosed herein,but that encode the same amino acid sequence with the same activity.

Functional equivalents encompass naturally occurring variants of thesequences described herein as well as synthetic nucleotide sequences,e.g., those nucleotide sequences that are obtained by chemical synthesisor recombination of naturally existing DNA. Functional equivalents maybe the result of, natural or synthetic substitutions, additions,deletions, replacements, or insertions of one or more nucleotides.

Examples of functional equivalents include those nucleic acid sequencescomprising a sense mutation resulting from the substitution of at leastone conserved amino acid which does not lead to an alteration in theactivity of the polypeptide, and thus can be considered functionallyneutral.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. For example, one exemplary guideline toselect conservative substitutions includes (original residue followed byexemplary substitution): ala/gly or ser; arg/lys; asn/gln or his;asp/glu; cys/ser; gln/asn; gly/asp; gly/ala or pro; his/asn or gln;ile/leu or val; leu/ile or val; lys/arg or gln or glu; met/leu or tyr orile; phe/met or leu or tyr; ser/thr; thr/ser; trp/tyr; tyr/trp or phe;val/ile or feu. An alternative exemplary guideline uses the followingsix groups, each containing amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (1); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W). Another alternative guideline is to allow for allcharged amino acids as conservative substitutions for each other whetherthey are positive or negative.

Individual substitutions, deletions or additions that alter, add ordelete a single amino acid or a small percentage (for example up to 26%,up to 20%, up to 10%, or up to 5%) of amino acids in an encoded sequenceare also considered to be functional equivalents.

Other non-limiting examples of functional equivalents include fragments,orthologs, splice variants, single nucleotide polymorphisims, andallelic variants.

Such functional equivalents will have 60%, 75%, 80%, 90%, 95% or greaterhomology to the nucleotide sequences disclosed herein.

Nucleotide sequence homology may be determined by sequence identity orby hybridisation.

Sequence identity may be determined using basic local alignment searchtool technology (hereinafter BLAST). BLAST technology is a heuristicsearch algorithm employed by the programs blastn which is available athttp://www.ncbi.nlm.nih.gov.

If homology is determined by hybridisation, the nucleotide sequencesshould be considered substantially homologous provided that they arecapable of selectively hybridizing to the nucleotide sequences disclosedherein.

Hybridisation should be carried out under stringent hybridisationconditions at a temperature of 42° C. in a solution consisting of 50%formamide, 5× standard sodium citrate (hereinafter SSC), and 1% sodiumdodecyl sulphate (hereinafter SDS). Washing may be carried out at 65° C.in a solution of 0.2×SSC and 0.1% SDS.

Background hybridization may occur because of other nucleotide sequencespresent, for example, in the cDNA. Any signal that is less than 10 foldas intense as the specific interaction observed with the target DNAshould be considered background. The intensity of interaction may bemeasured, for example, by radiolabelling the probe, e.g. with ³²P.

The nucleotide sequences may also comprise one or more of the following:a suitable 5′ untranslated region, a promoter to enable expression inappropriate host cells, a suitable 3′ untranslated region, a stop codonand tags.

Non limiting examples of tags include, but are not limited to, membraneexport tags and tags used for detection of the thioesterases including,but not limited to, His tag, glutathione-S-transferase tag (GST).

The 5′ untranslated region may comprise other operators or motifs thatinfluence the efficiency of transcription or translation. The 3′untranslated region may comprise other signals such as a signal fortranscriptional termination.

Non limiting examples of operators or motifs that influencetranscription or translation include, but are not limited to, signalsrequired for efficient polydenylation of the transcript, ribosomebinding sites, recognition sites e.g. EcoR1.

It is well within the purview of the person skilled in the art to selectsuitable 5′ and 3′ untranslated regions, tags, stop codons, andoperators or motifs that influence transcription or translation,depending on the host cells in question and the desired result. Nonlimiting examples are given in the examples included herein.

The nucleotide sequences disclosed in SEQ.ID. Nos. 1, 3, 5, and 7, orfunctional equivalents thereof, may be used to produce thioesteraseenzymes comprising the amino acid sequences disclosed in SEQ.ID. Nos. 2,4, 6, and 8, or functional equivalents thereof.

Expression of the enzymes with thioesterase activity comprising one ormore of the amino acid sequences, or functional equivalents thereof,disclosed in SEQ.ID. Nos. 2, 4, 6, and 8, may be effected by wellestablished cloning techniques.

According to another illustrative embodiment, there is provided a hostcell transfected with one or more of the nucleotide sequences disclosedin SEQ.ID. Nos. 1, 3, 5, and 7, or functional equivalents thereof. Suchhost cells are capable of expressing an enzyme with thioesteraseactivity comprising one or more of the amino acid sequences, orfunctional equivalents thereof, disclosed in SEQ.ID. Nos. 2, 4, 6, and8.

Suitable host cells include prokaryote and eukaryotic cells ofbacterial, fungal, plant or animal origin.

According to an illustrative embodiment the cells are bacterial orfungal cells.

In another illustrative embodiment the cells are selected from one ormore of Escherichia coli, Brevibacterium, Cornynebacterium,Arthrobacter, Pseudomonas, Nocardia, Methylobacteri, Lactobacillus,Lactococcus, Streptococcus, Pediococcus, Oenococcus, Leuconostoc,Weisella, Carnobacterium, and Tetragenococcus. Proprionibacterium sp.,Bifidobacterium spp., Enterococcus spp., Corynebacterium glutamicum,Arthrobacter sp., Micrococcus luteus and Staphylococcus equorum.Geotrichum candidum, Yarrowia lipolytica, Kluyveromyces lactis,Debaryomyces hansenii, Saccharomyces cerevisiae.

Host cells may be transfected with the nucleotide sequences, orfunctional equivalent thereof, transiently or stably, as is well knownin the art.

Any known method for introducing nucleotide sequences into host cellsmay be used. It is only necessary that the particular geneticengineering procedure used be capable of successfully introducing thedesired nucleotide sequences, or functional equivalents thereof, intothe host cell capable of expressing the desired amino acid sequences, orfunctional equivalents thereof. These methods may involve introducingcloned genomic DNA, cDNA, synthetic DNA or other foreign geneticmaterial into a host cell and include the use of calcium phosphatetransfection, polybrene, protoplast fusion, electroporation, liposomes,microinjection, expression vectors, and the like.

Expression vectors, both as individual expression vectors or aslibraries of expression vectors, comprising at least one nucleic acidsequences disclosed in SEQ.ID. Nos. 1, 3, 5, and 7, or functionalequivalent thereof, may be introduced and expressed in a cell's genome,a cell's cytoplasm, or a cell's nucleus by a variety of conventionaltechniques.

It is well within the purview of the person skilled in the art to selecta suitable technique.

According to an illustrative embodiment, expression vectors may be usedto transfect host cells with the nucleic acid sequences disclosed inSEQ.ID. Nos. 1, 3, 5, and 7, or functional equivalents thereof.

In another aspect of the present invention there is provided a vectorcomprising at least one nucleotide sequence disclosed in SEQ.ID. Nos. 1,3, 5, and 7, or functional equivalent thereof.

Any suitable expression vector may be used. Non limiting examples oftypes of vectors include bacteriophage, plasmid, or cosmid DNAexpression vectors; viral expression vectors, or bacterial expressionvectors.

It is well within the purview of the person skilled in the art to selecta suitable expression vector depending on the host cells in question andthe desired effect.

In an illustrative embodiment the vector is selected from pBL,pET-22b(+), Pgem-5z, pGIV1.

After transfection, the transfected cells may be cultured using standardculturing conditions well known in the art.

It will be apparent to the skilled person that different cells requiredifferent culture conditions including appropriate temperature and cellculture media. It is well within the purview of the person skilled inthe art to decide upon culture conditions depending on the cells inquestion and the desired end result.

In another aspect, there is provided a method of producing an enzymewith thioesterase activity comprising one or more of the amino acidsequences, or functional equivalents thereof, disclosed in SEQ.ID. Nos.2, 4, 6, and 8, comprising inserting a nucleotide sequence comprising atleast one nucleotide sequence disclosed in SEQ.ID. Nos. 1, 3, 5, and 7,or functional equivalent thereof, into a vector; transforming a hostcell with the vector and growing the transformed host cell in a suitableculture medium.

As stated above, it will be apparent to the skilled person thatdifferent cells require different culture conditions includingappropriate temperature and cell culture media. It is well within thepurview of the person skilled in the art to decide upon cultureconditions depending on the cells in question and the desired endresult.

Information on appropriate culturing media and conditions with respectto certain cells may be found on the American type culture collection(ATCC) Website:http://www.lgcstandards-atcc.org/Home/tabid/477/Default.aspx

In a particular illustrative embodiment the cells used wereBrevibacterium, Cornynebacterium or E Coli cells and the culture mediumwas dairy or whey based.

In another particular illustrative embodiment the cells used wereBrevibacterium, Cornynebacterium or E Coli cells and the culture mediumwas selected from tripticase-soy, de Man-Rogosa-Sharpe (MRS), Elliker's,M17, nutrient broth or LB medium. Cells were incubated overnight at 37°C.

In order to increase the yield of the thioesterase enzymes, thenucleotide sequences disclosed in SEQ.ID. Nos. 1, 3, 5, and 7, orfunctional equivalents thereof, may be over expressed by placing themunder the control of a strong constitutive promoter.

It is well within the purview of the person skilled in the art to selecta suitable constitutive promoter depending on the host cells and vectorin question.

If desired the thioesterase enzymes comprising the amino acid sequencesdisclosed in SEQ.ID. Nos. 2, 4, 6, and 8, or functional equivalentsthereof, may be isolated from the culture medium using methods wellknown in the art.

The isolated thioesterase enzymes comprising at least one of the aminoacid sequences disclosed in SEQ.ID. Nos. 2, 4, 6, and 8, or functionalequivalent thereof, or the transfected host cells comprising at leastone of the nucleotide sequences disclosed in SEQ.ID. Nos. 1, 3, 5 and 7,or functional equivalent thereof, may be used to produce thioesters.

In another aspect there is provided a method of producing thioesterscomprising contacting at least one thioesterase enzyme comprising atleast one amino acid sequences disclosed in SEQ.ID. Nos. 2, 4, 6, and 8,or functional equivalent thereof, and/or at least one host cellscomprising at least one of the nucleotide sequences disclosed in SEQ.ID.Nos. 1, 3, 5 and 7, or functional equivalent thereof, with at least onesuitable substrate, incubating the mixture, isolating the crude productcontaining the thioester and, purifying the crude product to obtain onlythe thioester.

Incubation may be carried out at a temperature of 20° C. to 40° C., at apH of 4 to 9, for a time period ranging from 1 to 100 hours.

In an illustrative embodiment incubation is carried out at a temperatureof 25° C. to 38° C., at a pH of 6 to 8, for a time period ranging from 1to 72 hours.

Short chain fatty acid coenzyme A derivatives have been found by theapplicant to be suitable substrates for the thioesterase enzymescomprising at least one of the amino acid sequences disclosed in SEQ.ID.Nos. 2, 4, 6, and 8, or functional equivalent thereof.

In an illustrative embodiment the suitable substrate comprises a shortchain fatty acid coenzyme A (SCFA-CoA).

In another illustrative embodiment a suitable substrate comprises aC1-C8 SCFA-CoA.

The transfected host cells and/or enzymes of the present invention maybe used in mobilised or immobilised form.

In an illustrative embodiment the enzymes and/or host cells are used inimmobilised form.

Any purification technique may used to purify the crude product and itis well within the purview of the person skilled in the art to decide onsuch a technique. Non limiting examples of purification techniquesinclude: affinity purification, centrifugation, chromatography.

In another aspect of present invention there is provided a thioesterobtainable or produced by a method as described herein.

In another aspect there is provided a kit for producing thioesterscomprising at least one thioesterase enzyme comprising at least oneamino acid sequence disclosed in SEQ.ID. Nos. 2, 4, 6, and 8, orfunctional equivalent thereof, and/or at least one host cells comprisingat least one of the nucleotide sequences disclosed in SEQ.ID. Nos. 1, 3,5 and 7, or functional equivalent thereof, and at least one suitablesubstrate.

The kit may be used to carry out the method, as herein disclosed, forproducing thioesters.

In an illustrative embodiment the suitable substrate comprises a shortchain fatty acid coenzyme A (SCFA-CoA).

In another illustrative embodiment the suitable substrate comprises aC1-C8 SCFA-CoA.

The C1-C8 SCFA-CoA substrate may be provided in a concentration from0.01 μM-500 μM, 0.01 μM-200 μM, 0.01 μM-50 μM.

It is known that the quantity and ratio of thioesters present in cheeseaffect its flavour. The thioesters are produced by microbes present inthe cheese during its manufacturing and ripening. These microbes areoften added to the cheese as starter cultures.

In another aspect there is provided a method of flavouring cheesecomprising adding to a cheese during its manufacture process at leastone thioesterase enzyme comprising at least one amino acid sequencesdisclosed in sequence ID numbers 2, 4, 6, and 8, or functionalequivalent thereof, and/or at least one host cells comprising at leastone of the nucleotide sequences disclosed in sequence ID numbers 1, 3, 5and 7, or functional equivalent thereof.

In an illustrative embodiment the thioesterase enzyme(s) and/or hostcell(s) are added as part of a starter culture.

In another illustrative embodiment the thioesterase enzyme(s) and/orhost cell(s) are added as part of a starter culture added to milk.

In another aspect there is provided a cheese product obtainable by orproduced according to the method herein above defined.

The nucleotide sequences disclosed in SEQ.ID. Nos. 1, 3, 5, and 7, orfunctional equivalents thereof, may be used to screen and identify wildtype organisms containing genes encoding the thioesterase enzymesencoded by the amino acid sequences disclosed in SEQ.ID. Nos. 2, 4, 6,and 8, or functional equivalents thereof.

In another aspect there is provided the use of the nucleotide sequencesdisclosed in SEQ.ID. Nos. 1, 3, 5, and 7, or functional equivalentsthereof, as markers in screening methods to screen for wild typeorganisms capable of producing the thioesterase enzymes encoded by theamino acid sequences disclosed in SEQ.ID. Nos. 2, 4, 6, and 8, orfunctional equivalents thereof.

In another aspect there is provided a method of identifying organismscapable of producing the thioesterase enzymes encoded by the amino acidsequences disclosed in SEQ.ID. Nos. 2, 4, 6, and 8, or functionalequivalents thereof, comprising using the nucleotide sequences disclosedin SEQ.ID. Nos. 1, 3, 5, and 7, or functional equivalents thereof, asmarkers in screening methods.

The organisms identified in the method may be any prokaryotic andeukaryotic organisms.

In an illustrative embodiment the organisms are wild type organisms.

Any known screening method may be used. Examples of well-known screeningmethods include, but are not limited to, computer driven nucleotide orprotein homology searches of public sequence databases, nucleotidescreening via high-throughput sequencing, and polymerase chain reaction(PCR) screening.

The thioesters formed by the methods disclosed herein may be isolatedand purified for use as flavouring compounds in many compositions andconsumable products.

In another aspect there is provided compositions comprising at least onethioester obtained or produced by the methods herein described.

In another aspect there is provided a method of creating, or modifyingthe flavour of a composition comprising adding to said composition, atleast one thioester formed as disclosed herein.

The thioesters may be present or added into a composition in neat formor in a solvent, or they may first be modified, for example byentrapment with an entrapment material such as for example polymers,capsules, microcapsules, nanocapsules, liposomes, precursors, filmformers, absorbents such as for example by using carbon zeolites, cyclicoligosaccharides and mixtures thereof, or they may be chemically boundto a substrates which are adapted to release the thioesters uponapplication of an exogenous stimulus such as light, enzyme, or the like.

One type of thioester may be the sole flavouring component of acomposition. Alternatively, multiple types of thioesters may be used incombination.

The composition may additionally comprise other flavourant ingredientsand excipients, for example carrier materials, conventionally used inflavour compositions.

Said other flavourant ingredients include, but are not limited to,natural flavours, artificial flavours, spices, seasonings, and the like.Exemplary flavouring ingredients include synthetic flavour oils andflavouring aromatics and/or oils, oleoresins, essences, distillates, andextracts derived from plants, leaves, flowers, fruits, and so forth, andcombinations comprising at least one of the foregoing.

Further examples of other flavourant ingredients can be found in“Chemicals Used in Food Processing”, publication 1274, pages 63-258, bythe National Academy of Sciences.

Said excipients conventionally used in flavour compositions include, butare not limited to, solvents (including water, alcohol, ethanol, oils,fats, vegetable oil, and miglyol), binders, diluents, disintegrantingagents, lubricants, flavouring agents, coloring agents, preservatives,antioxidants, emulsifiers, stabilisers, flavour-enhancers, anti-cakingagents, and the like.

Further examples of flavourant ingredients and excipients conventionallyused in flavour compositions may be found in “Perfume and FlavourMaterials of Natural Origin”, S. Arctander, Ed., Elizabeth, N.J., 1960;in “Perfume and Flavour Chemicals”, S. Arctander, Ed., Vol. I & II,Allured Publishing Corporation, Carol Stream, USA, 1994; in“Flavourings”, E. Ziegler and H. Ziegler (ed.), Wiley-VCH Weinheim,1998, and “CTFA Cosmetic Ingredient Handbook”, J. M. Nikitakis (ed.),1st ed., The Cosmetic, Toiletry and Fragrance Association, Inc.,Washington, 1988.

Other suitable and desirable ingredients of flavour compositions aredescribed in standard texts, such as “Handbook of Industrial ChemicalAdditives”, ed. M. and I. Ash, 2^(nd) Ed., (Synapse 2000).

The thioesters formed as disclosed herein may be used in compositions ata concentration of up to 100%. According to certain embodiments, thethioesters may be included in the compositions at a concentration in therange of about 0.01% to about 99%. According to other embodiments, thethioesters may be included in the compositions at a concentration in therange of about 1% to about 99%.

In another aspect there is provided a method of creating, enhancing ormodifying the flavour of a consumable product comprising adding to saidconsumable product at least one thioester formed as disclosed herein.

The thioesters formed as disclosed herein, or compositions containing atleast one thioester as disclosed herein, can be added to consumableproducts by using conventional techniques to directly admix saidthioesters or composition into the consumable product.

The quantities in which the thioesters formed as disclosed herein may beadded to consumable products may vary within wide limits and depend,inter alia, on the nature of the consumable product, on the effectdesired, the purpose of adding the thioesters formed as disclosedherein, to a consumable product, for example enhancing or creating ataste, and on the nature and quantity of any other components comprisedin the consumable product, for example other flavour ingredients. It iswell within the purview of the person skilled in the art to decide onsuitable quantities of the thioesters formed as disclosed herein toincorporate into a consumable product depending on the end use andeffect required.

Typical non-limiting concentrations, of the thioesters formed asdisclosed herein, in ppm by weight based on the weight of the consumableproduct, are about 500 ppm to about 0.01 ppm, more particularly about250 ppm to about 0.01 ppm, still more particularly about 100 ppm toabout 1 ppm.

In another aspect there is provided a consumer product comprising atleast one thioester obtained or produced by the methods hereindescribed.

The term consumable product(s) as used herein means any product that isintended to be placed in the oral cavity and ingested, or to be used inthe mouth and then discarded. Suitable consumable products include, butare not limited to, sauces, condiments, foodstuffs of all kinds,confectionery products, baked products, sweet products, savoury productsincluding meat flavoured and meat products and vegetable flavoured andvegetable products, dairy products, beverages, oral care products andcombinations thereof.

Exemplary foodstuffs include, but are not limited to, chilled snacks,sweet and savoury snacks, fruit snacks, chips/crisps, extruded snacks,tortilla/corn chips, popcorn, pretzels, nuts, other sweet and savourysnacks, snack bars, granola bars, breakfast bars, energy bars, fruitbars, other snack bars, meal replacement products, slimming products,convalescence drinks, ready meals, canned ready meals, frozen readymeals, dried ready meals, chilled ready meals, dinner mixes, frozenpizza, chilled pizza, soup, canned soup, dehydrated soup, instant soup,chilled soup, UHT soup, frozen soup, pasta, canned pasta, dried pasta,chilled/fresh pasta, noodles, plain noodles, instant noodles, cups/bowlinstant noodles, pouch instant noodles, chilled noodles, snack noodles,dried food, dessert mixes, sauces, dressings and condiments, herbs andspices, spreads, jams and preserves, honey, chocolate spreads, nut-basedspreads, and yeast-based spreads.

Exemplary confectionery products include, but are not limited to,chewing gum (which includes sugarised gum, sugar-free gum, functionalgum and bubble gum), centerfill confections, chocolate and otherchocolate confectionery, medicated confectionery, lozenges, tablets,pastilles, mints, standard mints, power mints, chewy candies, hardcandies, boiled candies, breath and other oral care films or strips,candy canes, lollipops, gummies, jellies, fudge, caramel, hard and softpanned goods, toffee, taffy, liquorice, gelatin candies, gum drops,jelly beans, nougats, fondants, combinations of one or more of theabove, and edible compositions incorporating one or more of the above.

Exemplary baked products include, but are not limited to, alfajores,bread, packaged/industrial bread, unpackaged/artisanal bread, pastries,cakes, packaged/industrial cakes, unpackaged/artisanal cakes, cookies,chocolate coated biscuits, sandwich biscuits, filled biscuits, savourybiscuits and crackers, bread substitutes,

Exemplary sweet products include, but are not limited to, breakfastcereals, ready-to-eat (“rte”) cereals, family breakfast cereals, flakes,muesli, other rte cereals, children's breakfast cereals, hot cereals,

Exemplary savoury products include, but are not limited to, salty snacks(potato chips, crisps, nuts, tortilla-tostada, pretzels, cheese snacks,corn snacks, potato-snacks, ready-to-eat popcorn, microwaveable popcorn,pork rinds, nuts, crackers, cracker snacks, breakfast cereals, meats,aspic, cured meats (ham, bacon), luncheon/breakfast meats (hotdogs, coldcuts, sausage), tomato products, margarine, peanut butter, soup (clear,canned, cream, instant, UHT), canned vegetables, pasta sauces.

Exemplary dairy products include, but are not limited to, ice cream,impulse ice cream, single portion dairy ice cream, single portion waterice cream, multi-pack dairy ice cream, multi-pack water ice cream,take-home ice cream, take-home dairy ice cream, ice cream desserts, bulkice cream, take-home water ice cream, frozen yoghurt, artisanal icecream, dairy products, milk, fresh/pasteurized milk, full fatfresh/pasteurized milk, semi skimmed fresh/pasteurized milk,long-life/UHT milk, full fat long life/UHT milk, semi skimmed longlife/UHT milk, fat-free long life/UHT milk, goat milk,condensed/evaporated milk, plain condensed/evaporated milk, flavoured,functional and other condensed milk, flavoured milk drinks, dairy onlyflavoured milk drinks, flavoured milk drinks with fruit juice, soy milk,sour milk drinks, fermented dairy drinks, coffee whiteners, powder milk,flavoured powder milk drinks, cream, yoghurt, plain/natural yoghurt,flavoured yoghurt, fruited yoghurt, probiotic yoghurt, drinking yoghurt,regular drinking yoghurt, probiotic drinking yoghurt, chilled andshelf-stable desserts, dairy-based desserts, soy-based desserts,

Exemplary beverages include, but are not limited to, flavoured water,soft drinks, fruit drinks, coffee-based drinks, tea-based drinks,juice-based drinks (includes fruit and vegetable), milk-based drinks,gel drinks, carbonated or non-carbonated drinks, powdered drinks,alcoholic or non-alcoholic drinks.

Thioesters are particularly important flavouring ingredients for dairyproducts, particularly cheese flavoured products. In particular, thethioester MTB, preferably used in combination with MTA and/or MTP, isdesired for providing an authentic cheese flavor with good flavorimpact.

In an illustrative embodiment the consumable product, comprising atleast one thioester formed as disclosed herein, is a cheese flavouredproduct.

In another illustrative embodiment the consumable product is a cheeseflavoured product comprising MTA, and/or MTB, and/or MTP formed asdisclosed herein.

SEQUENCE IDENTIFICATION

The invention has been described with reference to the followingsequence ID numbers:

SEQ ID No. 1—Depicts a nucleotide sequence encoding the thioesteraseenzyme 425.SEQ ID No. 2—Depicts an amino acid sequence of the thioesterase enzyme425.SEQ ID No. 3—Depicts a nucleotide sequence encoding one of thethioesterase 3320.SEQ ID No. 4—Depicts an amino acid sequence of the thioesterase enzyme3320.SEQ ID No. 5—Depicts a nucleotide sequence encoding the thioesteraseenzyme 1875.SEQ ID No. 6—Depicts an amino acid sequence of the thioesterase enzyme1875.SEQ ID No. 7—Depicts a nucleotide sequence encoding the thioesteraseenzyme 1874.SEQ ID No. 8—Depicts an amino acid sequence of the thioesterase enzyme1874.

The following examples are set forth to describe the invention furtherdetail and to illustrate the disclosed methods and products. However,the examples should not be construed as limiting in any manner.

EXAMPLES Example 1 Thioesterase Gene Cloning for Protein Expression

DNA sequences of thioesterase enzymes (hereinafter TE) TE 0425, TE 1875,TE 3320, and TE 1874 were obtained from B. linens strain ATCC9174 byamplification using polymerase chain reaction (PCR) with gene-specificprimers (Table 1).

The primers include restriction sites to allow directional cloning ofthe gene in the pET-22b(+) vector (Novagen), while avoiding sites thatoccur within the genes to be cloned. The 5′ primers include an XbaI sitefollowed by the sequence AGGAGGATTAACATATG, which provides a strong E.coli ribosome binding site and ATG start codon. After the ATG codon each5′ primer includes an in-frame gene-specific sequence of 14-20 basesstarting with the first base of the second codon of the open readingframe encoding the specific protein.

The 3′ primers includes a SalI or XhoI site and a gene-specific sequencecorresponding to the 17-23 base 3′ sequence of the specific open readingframe except for the native stop codon.

The pET-22b(+) vector is designed by Novagen to provide both inducibleexpression in E. coli and protein purification by the binding to nickelcolumns of a polyhistidine domain incorporated into the expressedprotein. Transcription of genes cloned into this vector is designed torequire the addition of the inducer IPTG, and use of the pET-22b(+)vector system for the purposes outlined in this invention was performedin full accordance to the manufacturer's recommendations.

For gene expression, E. coli BL21(DE3) host cells, which express the T7RNA polymerase required for the employed pET-22b(+) vector, were used.

Cloning of the resulting gene-specific XbaI to SalI or XhoI fragmentsinto the pET-22b(+) vector generated open reading frames with anin-frame sequence encoding a C-terminal His tag domain and a stop codonfrom the vector backbone.

TABLE 1 Gene-specific primers used to clone TE genes into the pET-22b(+) vector Gene 5′ Site 3′ Site Primers Used¹ TE 0425 XbaIGAATTCTAGAAGGAGGATTAACATATGTCCGTGAAGAC ATTCGAG SalIAGATCTGTCGACGCCGGCCTCGGCGCCGTATTC TE 1875 XbaICAATTGTCTAGAAGGAGGATTAACATATGTGTTCACGA TCCTATCGCGG XhoIAGATCTCTCGAGTCGGTTGCGACCCCGCATCGGT TE 3320 XbaIGAATTCTCTAGAAGGAGGATTAACATATGAATCCGCAG TCGGACGCA XhoIAGATCTCTCGAGGTTGGCCTCCTTCGGCATGGGGA TE 1874 XbaIGAATTCTAGAAGGAGGATTAACATATGAGCGATGCAGA ACCCACAG XhoIAGATCTCTCGAGCTGCGGAGCCTTCACACGGAC

Following polymerase chain reaction (PCR) amplification of thethioesterase genes with a mixture of the proofreading Pwo and Taqpolymerases (Expand 20 kb^(plus) PCR System, Roche Diagnostics GmbH),the PCR products were cloned without restriction digestion into thepGEM-5Z vector, using a pGEMT Vector System I cloning kit (PromegaCorporation).

After ligation to the vector DNA and electroporation, E. coli DH5αtransformant clones with the putative gene inserts were selected on LBagar plates containing 50 μg/mL ampicillin. The pGEM-5Z cloning allowedan initial screening of clones for inserts using the blue/whiteβ-galactosidase gene inactivation screen. Transformants were thenscreened for appropriately-sized plasmids by electrophoresis on 0.8%agaraose gels of plasmid DNA recovered from the ampicillin resistantcolonies.

In an alternative method to the above, the PCR products were cloneddirectly into pET-22b(+) after restriction digestion at the 5′ and 3′restriction sites included in the PCR primer sequences.

Plasmid DNAs from selected colonies were then digested with restrictionenzymes to confirm the presence of insert DNA with the predictedrestriction sites flanking the inserts and sizes using agarose gelelectrophoresis of the digestion products. Inserts passing these screenswere sequenced using primers binding to vector sites flanking theinserts. These were the SP6 and T7 promoter primers for pGEM-5Z clonesand the T7 promoter and T7 terminator primers for the pET-22b(+) clones.

Sequencing was done using big dye terminator methodology. Clones withthe expected sequences were identified in each case by perfectcomputer-based homology matches to their respective gene in the B.linens strain ATCC 9174 genome sequence.

For the sequences cloned initially in the pGEM-5Z vector, the insertswere recovered from appropriate restriction digestions by elution frompreparative agarose gels and then transferred into pET-22b(+) andscreened as described above. After obtaining the expected pET-22b(÷)constructs in E coli DH5α cells, the plasmids were recovered andtransformed by electroporation into E. coli BL21(DE3) cells for proteinexpression.

Example 2 Purification of the Cloned Thioesterases

Protein expression was induced in the E. coli BL21 (DE3) cells preparedin example 1 through the addition of 0.5 mM of IPTG to batch cultures(100 to 250 mL) comprising the aforementioned E. coli cells.

The E. coli cells were then incubated for 2-4 h, after which timepurification of the His-tagged proteins was performed under nativeconditions using the nickel-charged resin sold under the trade nameNi-NTA agarose (Invitrogen).

The incubated E. coli cells were then added to a solution comprising 50mM of Na-phosphate (pH 8.0), 20 mM of imidazole, 0.5 M of NaCl buffer,and disrupted by vortexing with glass beads.

The solution comprising the disrupted cells was then added to apurification column comprising 1.5 ml of NI-NTA agrose resin, preparedas directed by the manufacturer.

The cloned thioesterase proteins were extracted from the purificationcolumn with a buffer containing 50 mM of imidazole. After extraction theproteins were further eluted with 250 mM of the same buffer.

The extracted proteins were then dialyzed against 50 mM TrisHCl buffer(pH 8.0) at 4° C. The buffer was changed 4 times throughout the process.

Each thioesterase protein was then concentrated using 3K Amicon Ultra-4cartridges (Millipore, Inc., Billerica, Mass.) employed as directed bythe manufacturer.

The concentrations of each of the individual thioesterase proteins weredetermined using a micro-Lowry kit employed as directed by the kitsupplier (Sigma).

Example 3 SFCA's as Substrate

The ability of purified TE 425, TE 3320, TE 1875, and TE 1874 to producethioesters from fatty acids was determined. Unless stated otherwise theterm solution should be interpreted as being a water based solution.

A short chain fatty acid (hereinafter SCFA) mixture containing 10 mM ofeach of formic acid (C₁), acetic acid (C₂), propionic acid (C₃), butyricacid and iso-butyric acid (C₄), valeric acid and iso-valeric acid (C₅)and hexanoic acid (C₆) was prepared. This mixture was then diluted toyield a solution with a final total C₁ to C₆ SCFA concentration of 80μM.

The TE 425, TE 3320, TE 1875 and TE 1874 proteins purified as describedin example 2 were each separately diluted to yield separate solutionswith final enzyme concentrations of 0.35 μM.

Four sample solutions comprising 100 mM of phosphate buffer (pH 7.2),100 μM of bovine serum albumin (hereinafter BSA), 25 μM of methanethiol(hereinafter MeSH), 25 μM of coenzyme A (hereinafter CoASH), 2.5 μM of acomposition containing equal amounts of pyridoxal-phosphate,pyridoxamine and pyridoxal (hereinafter pyridoxal cocktail), 80 μM ofthe (C1-C6) SCFA mix prepared above, 10 μM of furfuryl alcohol, and 100μl of one of the four 0.35 μM enzyme solutions, were prepared.

Control samples were also prepared.

Control 1 was a solution comprising 100 mM of phosphate buffer (pH 7.2),100 μM of BSA, 25 μM of MeSH, 25 μM of CoASH, 2.5 μM of pyridoxalcocktail, 80 μM of the (C1-C6) SCFA mix, and 10 μM of furfuryl alcohol,but no enzyme.

Controls 2-5 were solutions comprising 100 mM of phosphate buffer (pH7.2), 100 μM of BSA, 25 μM of MeSH, 25 μM of CoASH, 2.5 μM of pyridoxalcocktail, 80 μM of the (C1-C6) SCFA mix, 10 μM of furfuryl alcohol, and100 μl of one of the four 0.35 μM enzyme solutions, but no (C1-C6) SCFAmix.

The contents of the samples and controls are illustrated in table 2.

TABLE 2 Samples (1-4) Control 1 (no enzyme) Controls 2-5 (no substrate)100 mM of 100 mM of 100 mM of phosphate buffer phosphate bufferphosphate buffer 100 μM of BSA 100 μM of BSA 100 μM of BSA 25 μM of MeSH25 μM of MeSH 25 uM of MeSH 25 μM of CoASH 25 μM of CoASH 25 μM of CoASH2.5 μM of pyridoxal 2.5 μM of pyridoxal 2.5 μM of pyridoxal cocktailcocktail cocktail 80 μM (C1-C6) 80 μM (C1-C6) SCFA mix SCFA mix 100 μlof one of the 100 μl of one of the four 0.35 μM four 0.35 μM enzymesolutions enzyme solutions 10 μM of furfuryl 10 μM of furfuryl 10 μM offurfuryl alcohol alcohol alcohol

After preparation all samples were then incubated at 37° C. for 35 min,with solid phase microextraction (SPME) carried out at the same time.

The 10 μM of furfuryl alcohol was added to the sample to correct forSPME variability.

For performing the SPME fibers coated with carboxen/polydimethylsiloxanewere used. The coating thickness was 85 mμm

The analytes absorbed or coated on the SPME fibres were then analysed bygas chromatography and mass spectrometry (GC-MS) at selected timeintervals.

None of the enzymes demonstrated thioester synthesis activity underthese assay conditions; i.e. no detectable production ofmethylthioacetate, methylthiopropionate, methylthiobutyrate, andmethylthiovalerate was recorded using the SCFA mix as a substrate.

Follow up experiments with single SCFAs as the substrate gave a similarnegative outcome.

Example 4 Coenzyme A-SFCAs Derivatives as Substrates

Since thioester synthesis was not detected from SCFA substrates, theability of purified TE 425, TE 3320, TE 1875, and TE 1874 to catalyzeMTB production from butyryl-coenzyme A (hereinafter butyryl-CoA) insteadof free butyrate was tested.

Four sample solutions were prepared by adding 5 μM of one of the fourthioesterase enzymes into a solution comprising 0.22 mM of BSA, 100 mMof phosphate buffer (pH 8.0), 0.03 mM butyryl-CoA, and 0.03 mM MeSH.

Six control samples were also prepared;

Control 7 was a solution comprising 0.22 mM of BSA, 100 mM of phosphatebuffer (pH 8.0), 0.03 mM butyryl-CoA, and 0.03 mM MeSH, but no enzyme.

Controls 8 to 11 were solutions comprising 0.22 mM of BSA, 100 mM ofphosphate buffer (pH 8.0), and 5 μM of the four thioesterase enzymes,but no butyryl-CoA.

Control 12 was a solution comprising 40 μM of MTB only. This was used asa control for SPME detection of MTB.

The contents of the samples and controls are illustrated in table 3.

TABLE 3 Samples 1-6 Control 7 Controls 8-11 Control 12 100 mM of 100 mMof 100 mM of phosphate phosphate phosphate buffer (pH 8.0) buffer (pH8.0) buffer (pH 8.0) 0.22 μM of BSA 0.22 μM of BSA 0.22 μM of BSA 0.03μM of 0.03 μM of 0.03 μM of MeSH MeSH MeSH 0.03 μM of 0.03 μM ofButyryl-CoA Butyryl-CoA 5 μm of one of the 5 μm of one of the fourenzymes four enzymes (TE425, TE 3320, (TE425, TE 3320, TE 1875, and TE1875, and TE1874) TE1874) 40 μM MTB

After preparation all samples were incubated at 32° C. After 2 hrs ofincubation SPME was carried out for 30 mins with the temperature kept at32° C. throughout the procedure. This resulted in a total incubationtime of 2.5 hrs.

10 μM of furfuryl alcohol was added to the sample to correct for SPMEvariability.

For performing the SPME fibers coated with carboxen/polydimethylsiloxanewere used. The coating thickness was 85 μm.

The analytes absorbed or coated on the SPME fibres were analysed usingGC-MS at selected time intervals. Results from this analysis are shownin table 4.

TABLE 4 MTB production from butyryl-CoA and MeSH by TE enzymes m/z 118Concentration of peak area MTB (μM) Control 7 0 0 Controls 8-11 0 0Control 12 62.02 40 Sample 1 TE425 2.96 1.908 Sample 2 TE 1874 1.561.006 Sample 3 TE 3320 11.73 7.57 Sample 4 TE 1875 6.15 3.966

Control 12 only contained MTB. This control indicated the expected m/zratio for MTB is 118.

Controls 7 to 12 did not show a peak at 118 and consequently it can beassumed that these samples do not contain any MTB (Table 3)

Samples 1, 2, 3, and 4 all show peaks at 118. This proves that in eachcase MTB has been formed and consequently that TE 425, TE 3320, TE 1875,and TE 1874 are thioesterase enzymes capable of producing thioestersfrom CoA-SCFA derivatives.

The amount of MTB produced by each enzyme can be estimated from the M/Z118 corresponding GC peak area that should be roughly proportional tothe amount of MTB in the sample. All peak areas and corresponding MTBconcentrations are listed in table 4.

Example 5 TE Enzymes Substrate Preferences and Product Profiles

The ability of purified TE 425 and TE 3320 to catalyze thioesterproduction from a mixture of SCFA CoA-derivatives was determined.

To investigate this activity, CoA-SFCA's were synthesized in a two stepreaction.

A preparation of fatty acid anhydrides was made via the followingmethod:

10 mmoles of N,N′-dicyclohexylcarbodiimide (DCC) was added to 50 mL ofdry carbon tetrachloride (CCl₄) (50 mL). This solution was then added toa second solution containing 20 mmoles of the SCFA mix in 150 mL of dryCCl₄.

The reaction mixture was kept at room temperature for 5 hrs after whichtime the dicyclohexylurea (DCU) precipitate was removed by filtration.Any CCL₄ in the precipitate was removed by evaporation under reducedpressure. The solid residue was re-crystallised from acetone. Thecrystals were washed with CCL₄ to remove traces of chemicals. Theproducts were then separated by chromatography to yield pure C₂ to C₈SCFA anhydrides.

After the SCFA anhydrides had been prepared and separated 30 μm of eachof the C₂ to C₈ SCFA anhydrides was added to a separate solution of 45μmol of 2.5 μM of pyridoxal cocktail in 30 mL of ice cold water.

Sodium bicarbonate was added to all the samples to ensure that in eachcase the pH was 7-7.5. All the mixtures were kept in an ice-bath andshaken frequently for 60 min.

The C₂ to C₈ SCFA-CoA's were purified by precipitation withtrichloroactetate (hereinafter TCA) and re-suspended in the water enzymeactivity assay.

Once the SOFA CoA's had been formed, samples comprising differentthioesterase and SFCA-CoA's were prepared by adding 35 μM of one of thethioesterase enzymes, either TE 425 or TE3320, plus 120 μM of the C₂ toC₈ SCFA-CoA mixture into a solution containing 100 μM of BSA, 100 mM ofphosphate buffer (pH 7.2), 25 μM of MeSH, and 2.5 μM of pyridoxalcocktail.

Control samples were also prepared;

Control 13 was a solution comprising 120 μM of the C₂ to C₈ SCFA-CoAmixture, 100 μM of BSA, 100 mM of phosphate buffer (pH 7.2), 25 μM ofmethanethiol, and 2.5 μM of pyridoxal cocktail, but no enzyme.

Controls 14 to 15 were solutions comprising 35 μM of one of thethioesterase enzymes, either TE 425 or TE3320, 100 μM of BSA, 100 mM ofphosphate buffer (pH 7.2), 25 μM of methanethiol, and 2.5 μM ofpyridoxal cocktail, but no C₂ to C₈ SCFA-CoA mixture.

The contents of the samples and controls are illustrated in table 5.

TABLE 5 Samples 1-2 Controls 13 Controls 14-15 120 μM of the C₂ to 120μM of the C₂ to C₈ SCFA-CoA C₈ SCFA-CoA 100 μM of BSA 100 μM of BSA 100μM of BSA 25 μM of MeSH 25 μM of MeSH 25 μM of MeSH 100 mM of phosphate100 mM of phosphate 100 mM of phosphate buffer (pH 7.2), buffer (pH7.2), buffer (pH 7.2), 2.5 μM of pyridoxal 2.5 μM of pyridoxal 2.5 μM ofpyridoxal cocktail cocktail cocktail 35 μM of one of the 35 μM of one ofthe thioesterase enzymes, thioesterase enzymes, either TE 425 or eitherTE 425 or TE3320 TE3320

All samples were incubated at 37° C. for 35 min, then SPME was carriedout for 30 mins with fibers coated (thickness=85 um) withcarboxen/polydimethylsiloxane. This resulted in a total incubation timeof 45 min.

10 μM of furfuryl alcohol was added to the sample to correct for SPMEvariability.

Analytes absorbed or coated on the SPME fibres were analysed by GC-MS atselected time intervals.

The results after incubation with equimolar mixture of SCFA-CoAderivatives, shown in table 6, confirm that TE 425 and 3320 havedifferent substrate selectivity.

TE 3320 showed a peak of activity for MTB and MTV formation (C4, C5);suggesting butyryl- and valeryl-CoA are preferred substrates. Incontrast, TE 425 gave a slight peak at MTH formation, suggestinghexanoyl-CoA (C6) may be its primary substrate.

TABLE 6 Enzymatic activity of TE 3320 and TE 425 with C₂ to C₈ SCFA-CoAmixture as substrates Enzyme activity (by initial velocity method)Thioester Product* MTA MTP MTB MTV MTH MTO Control 13 (no enzyme) 0.0 ±0.0  0.0 ± 0.0  0.0 ± 0.0 0.0 ± 0.0  0.0 ± 0.0  0.0 ± 0.0  Controls14-15 (no substrates) 0.2 ± 0.11 0.3 ± 0.15  0.2 ± 0.12 0.2 ± 0.12 0.1 ±0.06 0.1 ± 0.03 TE 3320 0.2 ± 0.11 0.1 ± 0.22 0.05 ± 0.05 0.1 ± 0.04 0.1± 0.05 0.1 ± 0.05 TE 425 0.2 ± 0.04 0.1 ± 0.06 0.12 ± 0.04 0.08 ± 0.05 0.1 ± 0.06 0.06 ± 0.02  *MTA, methylthioacetate; MTP,methylthiopropionate; MTB, methylthiobutyrate; MTV, methylthiovalerate;MTH, methylthiohexanoate; MTO, methylthiooctanoate

Example 6 Production of Various Thioesters by TE3320

A test sample was prepared by adding 100 μL of a solution comprising0.35 μM of TE 3320 to a solution comprising 100 mM of phosphate buffer(pH 7.2), 100 μM of BSA, 25 μM of methanethiol, 2.5 μM pyridoxalcocktail, and 4.16 μM of each of Acetyl-CoA (C2), Propionyl-CoA (C3),butyryl-CoA (C4), valeryl-CoA (C5), Hexanoyl-CoA (C6), and Octanoyl-CoA(C8).

Two control samples were also prepared.

Control 16 was a solution comprising 100 mM of phosphate buffer (pH7.2), 100 μM of BSA, 25 μM of methanethiol, 2.5 μM of pyridoxalcocktail, and 4.16 μM of each of Acetyl-CoA C2, Propionyl-CoA C3,butyryl-CoA C4, valeryl-CoA C5, Hexanoyl-CoA C6, and Octanoyl-CoA C8;but no enzyme.

Control 17 was a solution comprising 100 μL of a solution comprising0.35 μM of TE 3320, 100 mM of phosphate buffer (pH 7.2), 100 μM of BSA,25 μM of methanethiol, and 2.5 μM of pyridoxal cocktail; but no CoA-SFCAderivatives.

The contents of the samples and controls are illustrated in table 7.

TABLE 7 Sample Control 16 Control 17 100 mM of phosphate 100 mM ofphosphate 100 mM of phosphate buffer (pH 7.2) buffer (pH 7.2) buffer (pH7.2) 100 μM of BSA 100 μM of BSA 100 μM of BSA 25 μM of 25 μM of 25 μMof methanethiol methanethiol methanethiol 2.5 μM of pyridoxal 2.5 μM ofpyridoxal 2.6 μM of pyridoxal cocktail cocktail cocktail 4.16 uM of eachof 4.16 uM of each of Acetyl-CoA C2, Acetyl-CoA C2, Propionyl-CoA C3,Propionyl-CoA C3, butyryl-CoA C4, butyryl-CoA C4, valeryl-CoA C5,valeryl-CoA C5, Hexanoyl-CoA C6, Hexanoyl-CoA C6, and Octanoyl-CoA C8and Octanoyl-CoA C8 0.35 μM of TE 3320 0.35 μM of TE 3320

The samples were then incubated at 32° C. After 2 hrs of incubation SPMEwas carried out. The SPME took 30 mins and the temperature was kept at32° C. throughout the procedure. This resulted in a total incubationtime of 2.5 hrs.

10 μM of furfuryl alcohol was added to the sample to correct for SPMEvariability.

For performing the SPME fibers coated with carboxen/polydimethylsiloxanewere used. The coating thickness was 85 μm.

The analytes absorbed or coated on the SPME fibres were then analysedusing GC-MS.

The results from the GC-MS analysis are shown in table 8.

TABLE 8 Retention times, m/z fragment selection and TE 3320 enzymaticactivity of thioester formation. Sample TE 3320 Control 16 Control 17Thioester products Peak Area (No enzyme) (No substrate) MTA (C2) 1.3 0 090 m/z/7.9 min MTP (C3) 3.5 0 0 104 m/z/8.6 min MTB (C4) 4.7 0 0 118m/z/118; 9.3 min Methylthiovalerate (C5) 5.1 0 0 132 m/z/10.2 minMethythiohexanoate (C6) 3.2 0 0 146 m/z/11.12 min Methylthiooctanoate(C8) 3.4 0 0 174 m/z/13.32 min

Controls 16 and 17 both did not show peaks corresponding to anythioester products (Table 8). The sample containing the TE3320 enzymeshowed peaks at 7.9, 8.6, 9.3, 10.2, 11.12, and 13.32 mins with m/zvalues of 90, 104, 118, 132, 146, and 174, respectively.

This result proves that the thioesters were formed and that TE3320 is athioesterases capable of forming various thioesters from appropriatesubstrates.

The amount of each thioester produced by TE3320 can be estimated fromthe corresponding GC peak areas. Each peak corresponds to a particularthioester and the area of each peak should be roughly proportional tothe amount of each corresponding thioester in the sample. All peak areasare listed in table 8.

The larger peak area sizes at 9.3 and 10.2 min with m/z values of 118and 132, respectively, may indicate that TE3320 has a preference for C4and C5 substrates.

Example 7 Over Expression of B. linens Thioesterases

Over-expression of TE 425, TE 3320, TE 1875 and TE1874 in B. linens orother species of corynebacteria may be achieved via increased gene copynumber by inserting a nucleotide sequence of the present invention intoa suitable plasmid vector and transforming a host cell with said vector.

Suitable vectors include pGIV1, which is derived from the native plasmidpBLIN1 found in strain B. linens ATCC 9174. Features of pGIV1 includethe pBLIN1 replication origin and adjoining rep genes, the pUC originfor replication in E. coli, a replaceable ampicillin resistance geneflanked by multiple cloning sites where promoter-gene constructs can beinserted, and a gene for kanamycin resistance to facilitate selection inB. linens host cells.

Promoter-gene constructs for TE 425, 3320, 1875 and 1874 are comprisedof a suitable 5′ untranslated region, with operators or promoter motifsthat influence the efficiency of transcription or translation, plus theTE coding sequence.

Examples of suitable 5′ untranslated (“promoter”) regions include the200 bp region immediately upstream of REBL2645, a gene encodingacetolactate synthase large subunit, which may be strongly upregulatedby Met addition, as well as the 200 bp region immediately upstream ofRBLE02060, a gene encoding methionyl aminopeptidase that may beconstitutively expressed (Chalet et al. 2007, Appl. Microbiol.Biotechnol. 74:1320-1332).

Promoter-gene constructs were assembled following the general PCRstrategy described in example 1. The promoter fragments are 200 basepairs in length and flanked on their 5′ end by NotI sites and on their3′ end by Eco31I sites. In addition, the 3′ promoter primers wereconstructed to include an ATG start codon, a strong ribosome bindingsite (AGGAGG) starting ten nucleotides upstream of the start codon, andthe ‘consensus’ sequence CCAC between the ribosome binding site and thestart codon. The AGGAGG hexamer is complementary to a region near the 3′end of the B. linens 16 S ribosomal RNA sequence.

Thioesterase gene fragments are 603 base pairs for the TE 1875 and 435base pairs for TE 3320. Both are flanked on their 5′ ends by Eco31Isites and on their 3′ ends by HindIII sites. The 3′ end of thethioesterase gene fragment includes the TGA stop codon immediately 5′ tothe HindIII site.

These fragments were digested with the appropriate pairs of restrictionenzymes and then ligated in triple ligations into pBluescript(Stratagene, Agilent Technologies, Inc., Santa Clara, Calif.) that hadbeen digested with NotI and HindIII. The RBLE02060 and REBL2645 promoterfragments were ligated with the TE 1875 and TE 3320 gene insertfragments in the four pair wise promoter-gene combinations andtransformed into E. coli DH5 alpha cells.

The presence of correctly assembled promoter-gene combinations inplasmid DNA from prospective clones was confirmed by DNA sequenceanalysis (Table 9).

After sequence confirmation the insert fragments were recovered asNotI-HindIII restriction fragments for transfer into the B. linensshuttle vector pGIV1. The vector was digested in the polylinker regionwith the NotI and HindIII enzymes, then individual promoter-gene insertswere ligated into the vector and transformed into E. coli DH5 alphacells.

The presence of cloned promoter-gene combinations in recombinant plasmidDNA (pGIV1: TE plasmids) from prospective transformants was confirmed byDNA sequence analysis. Plasmid DNA was then isolated and transformedinto B. linens ATCC 19391 using the protocol described by Leret et al.1998 (Microbiol. 144:2827-2836) which is incorporated herein byreference.

Representative isolates of B. linens ATCC 19391 transformed with pGIV1:TE plasmids were selected, and over-expression of TE 425, 3320, 1875 or1874 may be confirmed by real-time quantitative PCR (Q-PCR) of TE mRNAtranscripts using primers based on the sequences provided in Table 1. Itis well within the purview of the person skilled in the art to designappropriate Q-PCR primers from such sequences and quantify cognate mRNAtranscripts.

TABLE 9 Nucleotide sequences for combined promoters and TE geneconstructs. Modified ribosome binding site and start codonregions are underlined and in bold font. promoters and TE geneconstructs Nucleotide sequences RBLE02060 promoter andCAGGCTCACCACGTCGCTGAGTCCGGAGAATTCGGGACCGGTCAC TE 1875:ATCTGCCCGTTCGCTCATCACTCCCCTTTGCTCAACATTGTGGCCTTCGCTGGCCGTTCCTTTCACTCTATTATCCTCTCCCACGGCAGGCCCGCACCGACAGGCACCGCAATTGTGGAATATCTGGGTTCAACCGC TCTAGAAT AGGAGGCACCATGTGTTCACGATCCTATCGCGGCCCT CACTGCAGCGGTGAAGGTGGCATGATGGGACATATGCACAACCGCACCACGAATCCCCATCTGAACGAATTCACACGAGTCCTGTTGGAACTGAGATGGGGAGACATGGATGCCTATGGCCACGTCAACAATGTCACCCAGCTGCGTCTGCTCGAAGAGGCACGCGTCCGCGCTCTGGGCTCACCGACGCACAGCACCGATGCACCCACAACTCCAGGTCAGCTGGGAATCTCAGGCACGGTATCGGGGATCACAATTCCGGCGATCTTCGCCGAGGCTTCGAACACCACCGAGCTGCTCGTCGCCTCCCACGCGATCGAATATCGCCGTCCCATTCCCTACCGTGCAGGTCCCATTGCCATCGATCTCGTCATCAGCGAGGTCAAACCGGCCTCTGTGACGATCGGTTACAGCATCGCCGAACCCGATGGTTCGGTCGGCTATACGCTGGCAGAGACGGTCATCGTCTTTGTAGACAGGACGACCTCCCGACCGCGTCGCCTGACCCAGGAGGAGACAGCAGCAATGGAAGACGTCATTCGACCTGCCGTACCGATGCGGGGTCGCAACCGATGA RBLE2645 promoter andGTCTCCCGTCTGATCGCCGGCAGCAACGAGATCCAGGAGATCGAG TE 1875:ATCAACCCGGTGCGCGTGACCCCGGATGGGGCATTGGCCGTCGACGCGCTCGTCGTCACGAACCGAGACGACAACGATGGCAGCACCGACAACGACAGCGGCAGCGACAACCCCGACAACGACAGCAGCACCG ACAACCCCGACA AGGAGGCACCATGTGTTCACGATCCTATCGCGG CCCTCACTGCAGCGGTGAAGGTGGCATGATGGGACATATGCACAACCGCACCACGAATCCCCATCTGAACGAATTCACACGAGTCCTGTTGGAACTGAGATGGGGAGACATGGATGCCTATGGCCACGTCAACAATGTCACCCAGCTGCGTCTGCTCGAAGAGGCACGCGTCCGCGCTCTGGGCTCACCGACGCACAGCACCGATGCACCCACAACTCCAGGTCAGCTGGGAATCTCAGGCACGGTATCGGGGATCACAATTCCGGCGATCTTCGCCGAGGCTTCGAACACCACCGAGCTGCTCGTCGCCTCCCACGCGATCGAATATCGCCGTCCCATTCCCTACCGTGCAGGTCCCATTGCCATCGATCTCGTCATCAGCGAGGTCAAACCGGCCTCTGTGACGATCGGTTACAGCATCGCCGAACCCGATGGTTCGGTCGGCTATACGCTGGCAGAGACGGTCATCGTCTTTGTAGACAGGACGACCTCCCGACCGCGTCGCCTGACCCAGGAGGAGACAGCAGCAATGGAAGACGTCATTCGACCTGCCGTACCGATGCGGGGTCGCAACCGATGA RBLE2060 promoter andCAGGCTCACCACGTCGCTGAGTCCGGAGAATTCGGGACCGGTCAC TE 3320:ATCTGCCCGTTCGCTCATCACTCCCCTTTGCTCAACATTGTGGCCTTCGCTGGCCGTTCCTTTCACTCTATTATCCTCTCCCACGGCAGGCCCGCACCGACAGGCACCGCAATTGTGGAATATCTGGGTTCAACCGC TCTAGAAT AGGAGGCACCATGAATCCGCAGTCGGACGCACTTCCA GATGTCTCACTTGCCTCAGCCAGCAACTTCGTCGCCGCCTCGGGGCTCGTCATCGACGAGGTCACGAACACAAGCGTCCGCGGCCATGCCGATCTGGGCAGCGACCACCACACGCCTTGGGGCGTCGTCCACGGCGGCGTGTACACAACGCTCGTGGAGAGCACGGGAAGCATTGGTGCCAGCCACGCTGTGGGCGAGCGCGGCGAGTTCGCCGTCGGCATCCACAACGCCACCGACTTTCTGCGCCCGACCGCCGGCGCCCGCGTTGCAGTCGAGGGCACCGCCCTGCATCAGGGCCGGACCCAGCAGCTGTGGGAGGTCATCATCACCGACACCTCATCGGACAAGGTCCTGGCCCGCGGCCAGCTGCGCCTGCAGAACGTCCCCATGCCGAAGGAGGC CAACTGARBLE2645 promoter and GTCTCCCGTCTGATCGCCGGCAGCAACGAGATCCAGGAGATCGAGTE3320: ATCAACCCGGTGCGCGTGACCCCGGATGGGGCATTGGCCGTCGACGCGCTCGTCGTCACGAACCGAGACGACAACGATGGCAGCACCGACAACGACAGCGGCAGCGACAACCCCGACAACGACAGCAGCACCG ACAACCCCGACA AGGAGGCACCATGAATCCGCAGTCGGACGCACT TCCAGATGTCTCACTTGCCTCAGCCAGCAACTTCGTCGCCGCCTCGGGGCTCGTCATCGACGAGGTCACGAACACAAGCGTCCGCGGCCATGCCGATCTGGGCAGCGACCACCACACGCCTTGGGGCGTCGTCCACGGCGGCGTGTACACAACGCTCGTGGAGAGCACGGGAAGCATTGGTGCCAGCCACGCTGTGGGCGAGCGCGGCGAGTTCGCCGTCGGCATCCACAACGCCACCGACTTTCTGCGCCCGACCGCCGGCGCCCGCGTTGCAGTCGAGGGCACCGCCCTGCATCAGGGCCGGACCCAGCAGCTGTGGGAGGTCATCATCACCGACACCTCATCGGACAAGGTCCTGGCCCGCGGCCAGCTGCGCCTGCAGAACGTCCCCATGCCGAAGG AGGCCAACTGA

Example 8 Heterologous Expression of Thioesterases

Heterologous expression of TE 425, TE 3320, TE 1875 and TE1874 inLactobacillus casei other lactic acid bacteria may be achieved viaincreased gene copy number by inserting a nucleotide sequence of thepresent invention into a suitable plasmid vector and transforming a hostcell with said vector.

Construction of thioesterase gene construct plasmids (hereinafter TEplasmids) for heterologous expression was performed by PCR following themethod described in example 1 except that a suitable vector containingsuitable 5′ untranslated region for heterologous expression of TE genesmust be used.

Suitable vectors are those derived from Lactobacillus casei or otherlactic acid bacteria and may include, but are not limited to, pHADH, aplasmid containing the cloned dhic gene from L. casei ATCC 334.(Broadbent et al. 2004, Appl. Environ. Microbiol. 70:4814-4820).

Suitable 5′ untranslated region for heterologous expression in pHADH maybe derived by PCR amplification of the putative ribosome binding siteand promoter region for dhic in using primers P1 and P2 (Table 10) toobtain a 285 bp XbaI/BglII fragment. The coding regions for TE 3320 orTE 1875 were amplified from B. linens ATCC9174 genomic DNA using primersB71 and B72 or B73 and B74, respectively.

These reactions yielded the complete TE3320 coding region on a 456 bpBglII/BamHI fragment, or the complete TE1875 coding region on a 609 bpBglII/BamHI fragment. The start codons were one (TE3320) or two (TE1875)basepairs downstream of the BglII site. No recognition sites for thesethree restriction enzymes (or SacI, see below) are present in the TEgenes or in the dhic promoter region.

TABLE 10 PCR primers used to clone TE genes into the pHADH-derived vector Primer Anneal Organism area Name Linker* SequenceL. casei dhic P1 XbaI GTTATCTAGACGATT ATCC promoter  TCTTATGGAGAG 334 5′dhic P2 BglII GTTTAGATCTCTTCC promoter  TTTCCAATTTGTCCA 3′ CTCACCAGB. linens TE3320 B71 BglII GTTTAGATCTCATGAATCC ATCC 5′ GCAGTCGGACGCACTTC9174 CAG TE3320 B72 BamHI GTTTGGATCCTC 3′ GAATCAGTGCCCAT CTCAGTTG TE1875B73 BglII GTTTAGATCTGCGTG 5′ TGTTCACGATCCTATCG CGG TE1875 B74 BamHIGTTTGGATCCAGTCA 3′ TCGGTTGCGACCC CGCATCGG *Restriction endonucleasetarget site embedded in primer sequence to facilitate directionalcloning.

The PCR fragments are digested with BglII and the promoter is separatelyligated to each coding sequence. The ligated DNA samples are recoveredand checked by PCR amplification with the primer combinations P1/B72 andP1/B74 (Table 10).

The promoter-TE coding region constructs were then digested with XbaIand BamHI and ligated into pBlueScript that was also digested with XbaIand BamHI and transformed into E. coli DH5 alpha. The presence ofcorrectly assembled promoter-gene combinations in transformants wasverified by PCR using primers P1×B72 (TE3320) and P1×B74 (TE1875) andDNA sequence analysis of insert regions from recombinant plasmids.

The recombinant plasmids are digested with SacI and BamHI and thepromoter-coding region fragments were separated from the pBlueScriptvector. The inserts were ligated into SacI/BamHI digested pHADH, whicheffectively replaces dhic with the promoter-TE coding region, andtransformed into E. coli DH5 alpha.

The presence of correctly assembled promoter-gene combinations intransformants was verified by PCR using primers P1×B72 (TE3320) andP1×B74 (TE1875) and DNA sequence analysis of insert regions fromrecombinant plasmids.

The recombinant plasmids pTE 3320 and pTE 1875 were recovered from E.coli clones and transformed by electroporation into L. casei ATCC 334 orother lactic acid bacteria.

For L. casei, one of several methods to transform cells begins byinoculating stationary-phase cells at 2% (vol/vol) into 500 mL of MRSbroth (Difco, Detroit, Mich., USA) and incubating at 37° C. until thesuspension reached an absorbance at 600 nm (A600) of 0.8.

The cells were harvested by centrifugation at 5000×g, washed twice withsterile, distilled water, and suspended in 2.5 ml. of ice-cold, sterile30% polyethylene glycol 1450 (Sigma Chemical Co.). 3 μl of the TEplasmid constructs formed above, are mixed with 100 μl of cellsuspension in a 0.2 cm electroporation cuvette and placed on ice for 3min. An electric pulse is delivered in a Bio-Rad Gene Pulser (Bio-RadLaboratories, Richmond, Calif., USA) set to the following parameters:2.5 kV, 25 μF, and 200Ω. After electroporation, 0.9 mL of warmed (37°C.) MRS broth was added, and the cells were incubated at 37° C. for 2 h.Transformed cells were collected on MRS agar containing 5 μg of ERY(Sigma Chemical Co., St. Louis, Mo., USA) per mL.

The presence of the TE vector in lysates from putative transformants wasconfirmed by electrophoretic separation in 0.6% agarose gels with Trisacetate buffer (40 mM Tris, 20 mM acetic acid, and 2 mM Na₂EDTA, pH8.1). It is well within the purview of the person skilled in the art toselect an appropriate lysis procedure for isolation of plasmid DNA,depending on the host cells in question.

The fidelity of promoter-gene combinations in pTE 3320 and pTE 1875transformants was verified by PCR using primers P1×B72 (TE3320) andP1×B74 (TE1875) and DNA sequence analysis of insert regions.

Overexpression of TE 425, 3320, 1875 or 1874 was confirmed by real-timequantitative PCR (Q-PCR) of TE mRNA transcripts using primers based onthe cognate gene sequences.

Alternatively, TE activity in these hosts can be demonstrated by enzymeassay for cell lysates using the method described in example 4.

While the nucleotide and amino acid sequences encoding thioesteraseenzymes, and various methods using and products incorporating the same,have been described in connection with various illustrative embodiments,it is to be understood that other similar embodiments may be used ormodifications and additions may be made to the described embodiments forperforming the same function disclosed herein without deviating therefrom. The embodiments described above are not necessarily in thealternative, as various embodiments may be combined to provide thedesired characteristics. Therefore, the disclosure should not be limitedto any single embodiment, but rather construed in breadth and scope inaccordance with the recitation of the appended claims.

1. A nucleotide sequence encoding an enzyme having thioesteraseactivity, said nucleotide sequence comprising of any one of thesequences disclosed in SEQ ID NOS 1 to 4 or functional equivalentsthereof.
 2. A vector comprising any one of the nucleotide sequences ofclaim
 1. 3. A host cell transfected with said vector of claim
 2. 4. Thehost cell of claim 3, wherein the host cell is of bacterial or fungalorigin.
 5. A method of producing an enzyme having thioesterase activitycomprising: inserting any one of the nucleotide sequences of claim 1into an expression vector; transfecting a host cell with said vector,and growing said transformed host cell in suitable culture conditions.6. A method of producing thioesters comprising contacting at least onethioesterase enzyme comprising at least one amino acid sequencedisclosed in SEQ ID NOS 2, 4, 6, and 8, or functional equivalentthereof, and/or at least one host cell comprising at least one of thenucleotide sequences disclosed in SEQ ID NOS 1, 3, 5 and 7, orfunctional equivalents thereof, with at least one suitable substrate,incubating the mixture, isolating the crude product containing thethioester and, purifying the crude product to obtain only the thioester.7. The method of claim 6, wherein the substrate comprises a C1-C8SCFA-CoA.
 8. A kit for producing thioesters comprising at least onethioesterase enzyme comprising at least one amino acid sequencedisclosed in SEQ ID NOS 2, 4, 6, and 8, or functional equivalentthereof, and/or at least one host cell comprising at least one of thenucleotide sequences disclosed in SEQ ID NOS 1, 3, 5 and 7, orfunctional equivalents thereof, and at least one suitable substrate. 9.A method of flavouring cheese comprising adding to a cheese during itsmanufacture process at least one thioesterase enzyme comprising at leastone amino acid sequence disclosed in SEQ ID NOS 2, 4, 6, and 8, orfunctional equivalent thereof, and/or at least one host cell comprisingat least one of the nucleotide sequences disclosed in SEQ ID NOS 1, 3, 5and 7, or functional equivalents thereof.
 10. The method of claim 9,wherein the thioesterase enzyme(s) and/or host cell(s) are added as partof a starter culture.
 11. A flavoured cheese product or a cheeseflavoured product obtainable by or produced by the method of claim 9.12. A method of screening for organisms capable of producing thioesterscomprising using any one of the nucleotide sequences shown in SEQ ID NOS1 to 4, or functional equivalents thereof, as markers.
 13. A compositioncomprising a thioester obtained by or produced by the method of claim 6.14. A consumable product comprising a thioester obtained by or producedby the method of claim
 6. 15. The consumable product according to claim10, wherein the consumable product is selected from the group consistingof: a cheese flavoured product or flavoured cheese product.
 16. Aflavoured cheese product or a cheese flavoured product obtainable by orproduced by the method of claim
 10. 17. A composition comprising athioester obtained by or produced by the method of any one of claim 7.18. A composition comprising a thioester obtained by or produced by themethod of any one of claim
 9. 19. A consumable product comprising athioester obtained by or produced by the method of claim
 7. 20. Aconsumable product comprising a thioester obtained by or produced by themethod of claim 9.